基于GRP78-ATF6通路探讨沉默SAT1对顺铂诱导耳蜗毛细胞损伤的影响

Investigating the effect of silencing SAT1 on cisplatin-induced damage to cochlear hair cells based on the GRP78-ATF6 pathway

摘要:

目的 探究精胺/亚精胺N［1］-乙酰基转移酶（SAT1）在顺铂诱导小鼠来源的永生化听觉毛细胞样细胞系（HEI-OC1）内质网应激中的作用机制，探讨SAT1对顺铂诱导耳蜗毛细胞损伤的影响。方法 将HEI-OC1细胞系按siNC、siSAT1、siNC+顺铂（30 μmol/L）、siSAT1+顺铂（30 μmol/L）分组，通过转染靶向SAT1的siRNA并联合顺铂处理，采用CCK-8法检测各组0、24、48、72 h细胞增殖活力；采用流式细胞术和透射电镜分析细胞凋亡率与超微结构变化；采用Western blotting法检测内质网应激标志物（IRE1α、XBP1s、GRP78、ATF6）及凋亡蛋白Caspase-12的表达。结果 4种SAT1-siRNA转染细胞24 h后均能沉默SAT1基因的表达，其中siSAT1-740组SAT1-siRNA的表达最低（P <0.05），因此选用siSAT1-740进行后续实验（表述为siSAT1）。Western blotting结果表明，siNC组的SAT1蛋白表达水平高于siSAT1组（P <0.05）。CCK-8实验结果表明，敲低SAT1能够显著缓解顺铂对HEI-OC1细胞的增殖抑制作用（P <0.05）。流式细胞术与透射电镜检测表明，敲低SAT1不仅能显著降低顺铂诱导的细胞早期、晚期及总凋亡率（P <0.05），还能从形态上明显改善染色质固缩与核碎裂等凋亡特征。Western blotting结果表明，各组细胞SAT1、IRE1α、XBP1s、GRP78、ATF6、Casepase-12蛋白表达比较，差异均有统计学意义（P <0.05）；在顺铂诱导的内质网应激条件下，沉默SAT1能够上调GRP78蛋白的表达（P <0.05）；下调IRE1α、XBP1s、ATF6、Caspase-12的表达（P <0.05）。结论 沉默SAT1基因后可能通过调控GRP78/ATF6通路，拮抗顺铂导致的细胞损伤，为药物性耳毒性的防治提供了潜在新靶点。

Abstract:

Objective To investigate the role of spermidine/spermine N1-acetyltransferase 1 (SAT1) in cisplatin-induced endoplasmic reticulum stress in mouse-derived immortalized auditory hair cell-like HEI-OC1 cells, and to define the impact of SAT1 on cisplatin-induced damage to cochlear hair cells.Methods HEI-OC1 cells were divided into four groups: siRNA negative control group (siNC), SAT1 gene silencing group (siSAT1), negative control with cisplatin treatment group (siNC + DDP), and SAT1 gene silencing with cisplatin treatment group (siSAT1 + DDP). The cells were transfected with SAT1-targeting siRNA and subsequently treated with cisplatin. Cell proliferation from 0 to 72 h was assessed using the CCK-8 assay; apoptosis and ultrastructural changes were analyzed by flow cytometry and transmission electron microscopy; endoplasmic reticulum stress markers (IRE1α, XBP1s, GRP78, ATF6) and apoptotic protein Caspase-12 were detected by Western blotting.Results All four SAT1-siRNA treatments effectively silenced SAT1 gene expression 24 hours post-transfection, with the most pronounced reduction observed in the siSAT1-740 group (P < 0.05). Consequently, the siSAT1-740 group was selected for subsequent experiments. Western blotting results showed that the protein expression level of SAT1 in the siNC group was significantly higher than that in the siSAT1 group (P < 0.05). Flow cytometry and transmission electron microscopy revealed that SAT1 knockdown not only significantly reduced DDP-induced early, late, and total apoptosis rates (P < 0.05), but also markedly improved apoptotic features such as chromatin condensation and nuclear fragmentation in morphological terms. Western blotting results indicate that the expression levels of SAT1, IRE1α, XBP1s, GRP78, Caspase-12, and ATF6 proteins exhibited statistically significant differences (P < 0.05) across different treatment groups. Under DDP-induced endoplasmic reticulum stress conditions, silencing SAT1 upregulates GRP78 protein expression (P < 0.05) while downregulating IRE1α, ATF6, Caspase-12 and XBP1s expression (P < 0.05).Conclusion The study revealed that silencing the SAT1 gene may counteract cisplatin-induced cellular damage by regulating the GRP78/ATF6 pathway, thus identifying SAT1 as a potential novel therapeutic target for preventing and treating drug-induced ototoxicity.CCK-8 assays demonstrated that SAT1 knockdown significantly alleviated the proliferation-inhibitory effect of DDP on HEI-OC1 cells (P < 0.05).